Wang Yi-Liang, Zhang Ying, Cai Da-Sheng
Department of Anaesthesiology, The First Hospital of China Medical University, Shenyang, 110001, PR China.
Department of Thyroid and Breast Surgery, Liaoning Provincial People's Hospital, Shenyang, 110001, PR China.
Cell Death Discov. 2022 Mar 8;8(1):105. doi: 10.1038/s41420-021-00784-7.
The purpose of the present study is to define the role of sevoflurane (SEV) in hepatic ischemia-reperfusion (I/R) injury as well as its underlying mechanism. Initially, hepatic I/R animal models and I/R hepatocyte models were established in C57BL/6 mice and normal mouse hepatocytes (BNL CL.2) after SEV preconditioning, respectively, followed by detection of microRNA-124-3p (miR-124-3p), TRAF3, and CREB expression by RT-qPCR and Western blot analysis. In addition, miR-124-3p, TRAF3 and CREB expression in hepatocytes was altered to identify their roles in modulating the levels of glutathione transferase (GST), aspartate aminotransferase (AST) and alanine aminotransferase (ALT), and inflammation-related factors and hepatocyte apoptosis by ELISA and flow cytometry respectively. The effects of SEV on the miR-124-3p/TRAF3/CREB axis were also verified in vitro and in vivo. IP assay was performed to verify the effect of TRAF3 on CREB ubiquitination in BNL CL.2 cells, and the cycloheximide (CHX) intervention experiment to detect the stability of CREB protein. SEV augmented the miR-124-3p expression in I/R animal and cell models. Moreover, SEV was observed to suppress I/R-induced liver damage, GST, ALT, and AST levels, hepatocyte apoptosis and inflammation. Overexpression of miR-124-3p resulted in alleviation of hepatic I/R injury, which was countered by TRAF3 overexpression. miR-124-3p targeted TRAF3, while TRAF3 promoted CREB ubiquitination and reduced protein stability of CREB. SEV could impede I/R-induced liver damage, GST, ALT, and AST levels, hepatocyte apoptosis and inflammation via mediation of the miR-124-3p/TRAF3/CREB axis in vitro and in vivo. Collectively, SEV may upregulate miR-124-3p to inhibit TRAF3 expression, thereby reducing the ubiquitination and degradation of CREB, alleviating hepatic I/R injury.
本研究的目的是确定七氟醚(SEV)在肝缺血再灌注(I/R)损伤中的作用及其潜在机制。首先,分别在七氟醚预处理后的C57BL/6小鼠和正常小鼠肝细胞(BNL CL.2)中建立肝I/R动物模型和I/R肝细胞模型,随后通过RT-qPCR和蛋白质免疫印迹分析检测微小RNA-124-3p(miR-124-3p)、肿瘤坏死因子受体相关因子3(TRAF3)和环磷腺苷效应元件结合蛋白(CREB)的表达。此外,改变肝细胞中miR-124-3p、TRAF3和CREB的表达,以分别通过酶联免疫吸附测定(ELISA)和流式细胞术确定它们在调节谷胱甘肽转移酶(GST)、天冬氨酸氨基转移酶(AST)和丙氨酸氨基转移酶(ALT)水平以及炎症相关因子和肝细胞凋亡中的作用。还在体外和体内验证了SEV对miR-124-3p/TRAF3/CREB轴的影响。进行免疫沉淀(IP)试验以验证TRAF3对BNL CL.2细胞中CREB泛素化的影响,并进行环己酰亚胺(CHX)干预实验以检测CREB蛋白的稳定性。SEV增加了I/R动物和细胞模型中miR-124-3p的表达。此外,观察到SEV可抑制I/R诱导的肝损伤、GST、ALT和AST水平、肝细胞凋亡和炎症。miR-124-3p的过表达导致肝I/R损伤减轻,而TRAF3的过表达则抵消了这种作用。miR-124-3p靶向TRAF3,而TRAF3促进CREB泛素化并降低CREB的蛋白质稳定性。SEV可通过在体外和体内介导miR-124-3p/TRAF3/CREB轴来减轻I/R诱导的肝损伤、GST、ALT和AST水平、肝细胞凋亡和炎症。总体而言,SEV可能上调miR-124-3p以抑制TRAF3表达,从而减少CREB的泛素化和降解,减轻肝I/R损伤。